Stimulation of monovalent ion fluxes and DNA synthesis in 3T3 cells by melittin and vasopressin is not mediated by phospholipid deacylation

Melittin at subtoxic concentrations stimulates monovalent ion fluxes and, together with insulin, synergistically increases DNA synthesis, but has little effect on phospholipase activity. The Na⁺ ionophore monensin increases Na-K pump activity without affecting phospholipase activity; whereas, the divalent cation ionophore A23187 has reciprocal effects. Finally, vasopressin and insulin potently stimulate ion fluxes and DNA synthesis but have no effect on phospholipase activity. Thus, the polypeptide mitogens melittin, vasopressin, and insulin alter membrane function and subsequently stimulate DNA synthesis in quiescent 3T3 cells by mechanisms independent of phospholipid deacylation.     

Serum stimulates na entry and the Na-K pump in quiescent cultures of epithelial cells (MDCK)

The growth of an epithelial canine kidney line (MDCK) was reversibly arrested by gradually lowering the serum concentration in the medium over a 3-day period. The cells were demonstrably quiescent by autoradiography after an additional 24 hours in serum-free media. Addition of fresh serum produced DNA synthesis after an 18-hour lag period. The quiescent cells then grew to confluency retaining their transport capacities as seen by the formation of “domes”. This system allows for measurement of monovalent ion fluxes and its relationship to growth regulation. The addition of fresh serum to quiescent MDCK cells increased the uptake of ⁸⁶Rb, a measure of Na-K pump activity. This stimulation was mediated by increased uptake of Na into the cells. Serum-stimulated DNA synthesis was blocked by the addition of ouabain in concentrations that inhibit the Na-K pump. Serum appears to stimulate growth in epithelial cells by increasing the amount of intracellular Na available to the Na-K pump. Monovalent ion transport may play a role in the regulation of epithelial cell proliferation.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Vasopressin rapidly stimulates NA entry and NA-K pump activity in quiescent cultures of mouse 3T3 cells

Addition of (Arg) vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates an ouabain-sensitive ⁸⁶Rb uptake. In contrast the hormone has no significant effect on the rate of efflux of this cation from preloaded cells. The stimulation of ⁸⁶Rb uptake is cycloheximide-insensitive, occurs within minutes of hormone addition and results from an increase in the Vmax of the uptake system. Vasopressin stimulates ion uptake in a concentration-dependent fashion (1-100 ng/ml); oxytocin also stimulated the Na-K pump but at significantly higher concentrations. The stimulation of the Na-K pump by vasopressin is apparently mediated by an increase in Na entry into the cells, since the hormone (1) strikingly shifts the concentration dependence on Na⁺ of the Na-K pump, (2) increases ²²Na uptake, and (3) increases intracellular Na contents when the efflux of this ion is blocked by ouabain. Since vasopressin is a potent mitogen for Swiss 3T3 cells, the results provide further evidence in support of a possible role of monovalent ion fluxes in signalling the initiation of growth stimulation.     

Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation

In addition to its cytotoxic/cytostatic action on many tumor cells in vitro, tumor necrosis factor (TNF) was recently shown to stimulate the growth of some types of cells in culture. We examined the action of TNF in BALB/c 3T3 cells which have been used extensively to study growth regulation. In subconfluent, rapidly dividing 3T3 cultures, murine (Mu) TNF was cytotoxic, while human (Hu) TNF had virtually no antiproliferative action on the cells. In contrast, in density-arrested BALB/c 3T3 cells maintained in a chemically defined, serum-free medium, MuTNF produced a dose-dependent stimulation of DNA synthesis. In stimulating DNA synthesis, MuTNF acted synergistically with both epidermal growth factor or platelet-derived growth factor. While stimulating DNA synthesis in quiescent 3T3 cultures, high doses of MuTNF (100 or 10 ng/ml) were also cytotoxic for a portion of the cells in the same cultures. Cytotoxicity was apparent 2 h after the addition of MuTNF, well before the onset of DNA synthesis. BALB/c 3T3 cell variants selected for their resistance to the cytotoxic action of MuTNF retained the capacity to respond to the mitogenic action of MuTNF, indicating that the stimulation of DNA synthesis by TNF is not a consequence of a TNF "wounding effect." Addition of TNF to density-arrested 3T3 cells resulted in the release of free arachidonic acid and palmitic acid from the cells. Quinacrine, a phospholipase inhibitor, inhibited both cytotoxicity and DNA synthesis in response to TNF, and melittin, a phospholipase activator, mimicked both the cytotoxic and mitogenic actions of TNF in quiescent BALB/c 3T3 cells. These results suggest that phospholipid breakdown is part of the essential early signal transduction events required both for the cytotoxic and mitogenic response to TNF action.     

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.     

Increases in phosphatidic acid levels accompany sphingosine-stimulated proliferation of quiescent Swiss 3T3 cells

Sphingosine, a breakdown product of cellular sphingolipids, has recently been shown to stimulate DNA synthesis and act synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts (Hong, Z., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in cytosolic phosphatidic acid, which is a potent mitogen for Swiss 3T3 cells. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, did not mimic the mitogenic effect of sphingosine and did not elevate phosphatidic acid levels. Sphingosine not only stimulated [3H]thymidine incorporation with similar efficiency and kinetics as phosphatidic acid, it also induced similar morphological alterations. Both sphingosine and phosphatidic acid acted synergistically with a variety of growth factors, such as, insulin, epidermal growth factor, fibroblast growth factor, and 12-O-tetradecanoylphorbol 13-acetate. In sharp contrast, sphingosine and phosphatidic acid did not have additive or synergistic effects in either the presence or absence of other growth factors. Both sphingosine and phosphatidic acid stimulated DNA synthesis in cells made protein kinase C-deficient by prolonged treatment with phorbol ester and sphingosine still stimulated similar increases in phosphtidic acid in these cells. Furthermore, similar to the actions of phosphatidic acid on signal transduction in Swiss 3T3 cells, mitogenic concentrations of sphingosine also inhibit cAMP accumulation and trigger the hydrolysis of polyphosphoinositides. Our findings indicate that sphingosine and phosphatidic acid control cellular responses in Swiss 3T3 cells through a common pathway. In view of the prominent role of phosphatidic acid in signal transduction and cellular proliferation, our observations that sphingosine, at mitogenic concentrations, increases the level of phosphatidic acid and also mimics the effects of phosphatidic acid on signal transduction, have important implications for the mechanism of action of sphingosine.     

Na entry and Na-K pump activity in murine,hamster, and human cells - effect of monensin,serum, platelet extract,and viral transformation

The relationship between Na entry and the activity of the Na-K pump has been investigated in a variety of cell types by testing the effect of the Naionophore monensin, mitogenic stimulation with serum and oncogenic transformation by SV₄₀ and polyoma virus. We found that addition of monensin increases intracellular Na in quiescent cultures of murine, hamster, and human cells. In each case, the rise in intracellular Na by monensin is associated with an increase in the activity of the Na-K pump, which was measured as ouabain-inhibitable ⁸⁶Rb uptake. The addition of serum to quiescent cultures stimulates ⁸⁶Rb uptake in all cell types studied. Serum alone causes an increase in intracellular potassium with no consistent change in intracellular Na. In the presence of the Na-K pump inhibitor ouabain, serum causes a marked increase in intracellular Na, with little change in intracellular K. This pattern is interpreted as indicating that the primary effect of serum is to increase Na entry into the cells. A low concentration of monensin (0.2 μg/ml) mimics the effect of serum on ion fluxes and content, which supports the conclusion that serum and monensin stimulate ⁸⁶Rb uptake in the same manner, namely by increasing Na entry into the cells. In addition, a partially purified platelet extract stimulates Na entry and ⁸⁶Rb uptake in quiescent 3T3 cells. Finally 3T3 cells transformed by SV₄₀ or polyoma virus exhibit a higher rate of Na entry and of Na-K pump activity than their untransformed 3T3 counterparts. All these results indicate that the rate of Na entry plays an important role in the regulation of the activity of the Na-K pump and that an increase in Na and K movements is a rapid response elicited by serum in a variety of cell types.     

Effect of heparin-binding growth factors on monovalent cation transport in Balb/C 3T3 cells

The changes in monovalent cation fluxes induced by class 1 and class 2 heparin-binding growth factors purified from bovine brain were studied in confluent quiescent Balb/C 3T3 cells. Both classes of HBGF's rapidly stimulate the activity of the Na+-K+ pump in a concentration-dependent manner that parallels their mitogenic activity. This effect requires the presence of external Na+ and is abolished by 1 mM amiloride. As with other growth factors, HBGF's increase the activity of the Na+-K+ pump by increasing Na+ influx into the cells via the amiloride-sensitive Na+-H+ exchange. These results indicate that rapid activation of cation transport is an early cellular response that follows binding of HBGF's to specific receptors on the surface of target cells.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

Glycolysis in quiescent cultures of 3T3 cells. Stimulation by serum, epidermal growth factor, and insulin in intact cells and persistence of the stimulation after cell homogenization.

Addition of serum to quiescent cultures of 3T3 cells rapidly increases lactic acid formation and subsequently stimulates cell division. The stimulation of lactic acid production is seen at high, saturating concentrations of extra-cellular glucose. It is dependent on the time of exposure and on the dose of serum and is not blocked by the addition of cycloheximide, puromycin, or actinomycin D. In contrast, serum only marginally affects glycolysis by rapidly growing 3T6 or SV40-3T3 cells. In addition to serum, epidermal growth factor (0.1 to 10 ng/ml) and insulin (10 to 500 ng/ml) cause a striking stimulation of glycolysis in quiescent 3T3 cells. Neither exogenous cyclic nucleotides nor ouabain effect the glycolytic response, but the presence of Ca2+ markedly influences the activation of glycolysis by epidermal growth factor and by insulin. A novel finding in this study is that homogenates prepared from quiescent cells treated with serum, epidermal growth factor, or insulin show increased glycolysis as compared with homogenates from nonstimulated cultures. This finding will allow further experimental analysis of the cause of increased glycolysis in rapidly proliferating cells.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Cyclic AMP stimulation of Na-K pump activity in quiescent swiss 3T3 cells

Recently, we have found that an increase in the intracellular level of cAMP acts as a mitogenic signal for Swiss 3T3 cells (Rozengurt et al., Proc. Natl. Acad, Sci. USA, 78:4392, 1981). The results presented in this paper demonstrate that addition of cAMP-elevating agents to confluent and quiescent cultures of Swiss 3T# causes a marked increase in the rate of 86Rb+ uptake but has no effect on the rate of cation efflux. The stimulation of ion uptake is mediated by the Na-K pump as shown by the ouabain sensitivity of the 86Rb+ fluxes. The increase in Na-K pump activity occurs whether cAMP is generated endogenously by stimulation of adenylate cyclase activity by cholera toxin, adenosine agonists, or PGE1 or added exogenously as 8BrcAMP. The stimulatory effect of these compounds on 86Rb+ uptake is potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity. Cholera toxin stimulates the Na-K pump in a dose-dependent manner; half-maximal effect is achieved at 0.7 ng/ml. The stimulation of ouabain-sensitive 86Rb+ uptake by cAMP-elevating agents reaches a maximum after 2-3 h of incubation. This contrasts with the rapid (within minutes) stimulation of the Na-K pump caused by serum and other mitogenic agents. Further, cAMP-elevating agents fail to increase Na+ influx into 3T3 cells whereas serum causes a marked increase in Na+ influx, under identical experimental conditions. These findings suggest that the stimulation of Na-K pump activity caused by increased cAMP levels contrasts mechanistically with the rapid control of pump activity by serum which is primarily mediated by increased Na+ entry into the cells.     

Evidence for early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Regulation of transmembrane signal transduction of insulin-like growth factor II by competence type growth factors or viral ras p21

In BALB/c 3T3 cells pretreated with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (primed-competent cells), insulin-like growth factors I and II (IGF-I and IGF-II) bind to their own receptors (IGF-IR and IGF-IIR) and stimulate calcium influx and DNA synthesis by a mechanism involving a 40-kDa pertussis toxin substrate. In contrast, these IGFs do not act on unprimed quiescent cells. In this study, the 40-kDa pertussis toxin substrate was identified as Gi-2 alpha using anti-G protein antibodies. We analyzed the quality of signal transduction from IGF-II to Gi-2 alpha. There was no difference in the amount of Gi-2 alpha between quiescent and primed-competent cells, and both of these cells had similar Kd values and numbers of IGF-II-binding sites. Whereas IGF-II did not alter pertussis toxin-catalyzed ADP-ribosylation of Gi-2 alpha in quiescent cells, IGF-II reduced the pertussis toxin substrate activity by 35-50% via the IGF-IIR in primed-competent cells. The action of IGF-II lasted for up to 3 h when IGF-II was present in the medium, and it disappeared when IGF-II was removed. These results suggest that the signaling pathway triggered by IGF-II is uncoupled between the IGF-IIR and Gi-2 alpha in quiescent cells and that PDGF and EGF restore the IGF-IIR-Gi-2 coupling. This study also indicates that low concentrations of IGF-I reduce the pertussis toxin substrate activity of Gi-2 alpha in primed-competent cells in a time course slower than that of IGF-II, but not at all in quiescent cells. However, both of these cells had similar Kd values and numbers of IGF-I binding sites. Therefore, the IGF-I signaling pathway may also be uncoupled between the IGF-IR and Gi-2 alpha in quiescent cells and restored by PDGF and EGF. In BALB/c 3T3 cells transfected with temperature-sensitive Kirsten sarcoma virus bearing the v-Ki-ras gene (ts cells), a 40-kDa pertussis toxin substrate was also identified as Gi-2 alpha. In nonpermissive ts cells, IGF-II was without effect on the pertussis toxin substrate activity of Gi-2 alpha or on calcium influx.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sphingomyelinase and cell-permeable ceramide analogs stimulate cellular proliferation in quiescent Swiss 3T3 fibroblasts.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.